Induction of immunological tolerance

ABSTRACT

A method of creating tolerance to transplanted cells, tissue, or organs without the need for continuous immunosuppression. A tolerizing dose of a cell or tissue within a membrane structure is implanted into a patient. Once the patient becomes tolerant to the cell or tissue, a tissue or organ is implanted which will no longer be recognized as foreign matter. The method makes animal organs practical for human use, prevents autoimmune destruction as well as immune rejection. It has applications in treatment and prevention of many mammalian diseases.

RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.10/660,924 filed Sep. 12, 2003 which is a continuation of applicationSer. No. 09/226,742 filed Jan. 7, 1999 now abandoned which was acontinuation of application Ser. No. 09/049,757 filed Mar. 27, 1998 nowabandoned, which was a continuation of application Ser. No. 08/736,413filed on Oct. 24, 1996 now abandoned, which claims the benefit ofpriority under 35 U.S.C. 119(e) of Provisional Application No.60/005,877 filed Oct. 26, 1995.

FIELD OF THE INVENTION

[0002] The present invention relates to the induction of immunologicaltolerance to foreign cells, tissues and organs. More specifically, theinvention relates to implantation of a tolerizing dose of cells ortissues encapsulated in a membrane in a mammal to establishimmunological tolerance thereto.

BACKGROUND OF THE INVENTION

[0003] For some human diseases, including heart and liver failure, organtransplantation is the only alternative to certain death. While therewere only 4,843. organ donors in the U.S. in 1993, there were 2,866heart and 3,040 liver failure patients on the waiting list for theseorgans (UNOS Update, 10(2), 1994). Thus, because of timing and tissuematching problems, many patients die each year for lack of an availableorgan. For those lucky enough to receive an organ, the results are stillless than ideal. The transplant procedure constitutes major surgerywhich is associated with attendant risks and is exceedingly expensive.After the surgery, the patient must be placed on a regimen ofimmunosuppressive drugs to keep the immune system from destroying thetransplanted organ. As a consequence, the patient's entire immune systemis suppressed for the rest of his life, significantly lowering hisdefenses against other serious disease threats such as infections,viruses or cancers.

[0004] For other diseases including kidney failure, pancreas failure andcystic fibrosis, transplantation has a lower mortality and morbidityrate than any alternative therapy. Even with its attendant problems oforgan scarcity, surgical risk, high cost and permanentimmunosuppression, for some of these cases it is still a more practicaltherapy than any alternative. The physician's choice in these cases isdependent on many variables including age, general health, severity ofthe condition, availability of organs and other factors. In 1994, therewere 25,033 patients on the waiting list for human kidneys, 181 forpancreases and 1,250 for lungs (UNOS Update, 10:2, 1994).

[0005] For still other diseases, transplantation is known to beeffective, although its attendant problems preclude its practicaltherapeutic use. This is true for many of the kidney, pancreas and lungpatients described above. It is also true where whole pancreastransplantation can cure diabetes or liver transplantation can curehemophilia but the risks outweigh the rewards.

[0006] Recently, for certain disease states; tissue transplants, asopposed to whole organ transplants, have been shown to be therapeutic inanimals and even in man (Scharp et al., Transplantation, 51:76-85,1991). Tissue transplantation requires full immunosuppression andcarries the same risks and problems as already discussed for whole organimmunosuppression. The following treatments address the rejection of thetransplanted tissue.

[0007] One implantation method involves pre-inoculation in the thymuswith a small dose of cells, full temporary immunosuppression, then afull therapeutic dose at another site (Posselt et al., Annals ofSurgery, 214:363-373, 1991). First, this has only been shown to work inrodents to date. No large animal or human test has been successful.Second, the human adult thymus is shrunken and may not be practical totreat with an adequate pre-dose. Third, the immunosuppression step,while temporary, does subject the patient to risks for that period oftime. Fourth, it is not known whether a fully therapeutic dose will betolerated, (i.e. not rejected) in humans. Fifth, this procedure may notprotect against autoimmune destruction even if it does preventrejection.

[0008] Another method of preventing rejection is irradiation of therecipient's bone marrow immune cells, implantation of bone marrow cellsfrom the donor, then implantation of a full therapeutic dose of tissueor organ from the same donor (Illstad et al., J. Exp. Med., 174:467-478,1991). First, this has not been shown to work for tissue transplants inhumans. Second, irradiation of immune cells, either partial or wholebody, carries serious risks. Third, it is not known if the immune systemwill adequately protect from other threats. Fourth, it is not known ifthe method will protect from both rejection and autoimmune destructionin those disease states.

[0009] A further method of treatment to prevent rejection is by usingmonoclonal antibodies to suppress certain parts of the immune system(Andersson et al., J. Autoimmun., 4:733-742, 1991). These tests haveonly been performed in rodents so it is not known if they would succeedin humans. Also, it is not known if the proper monoclonal antibody couldbe identified and created for each different disease state. In addition,the overall affect of these agents on the human immune system is notknown.

[0010] Still another method of preventing rejection is encapsulation ofthe transplanted tissue in a semi-permeable membrane device which allowsoxygen, nutrients and other small molecules to pass but prevents entryof large immune system cells (Lacy et al., Science, 254:1782-1784, 1991;Sullivan et al., Science, 252:718-721, 1991). There are severalunresolved problems associated with this method. First, none of thesedevices has been shown to protect a therapeutic transplant in humans. Tobe suitable for human use, the material must be biocompatible; it mustbe sufficiently strong to last a long time when implanted; its porositymust be exactly correct to allow survival and function of the enclosedcells while keeping out cells and perhaps antibodies of the immunesystem; and finally, the device itself must be large enough to containenough cells for a fully therapeutic implant and yet small enough toallow for some reasonable method of implantation which causes no damageto other internal organs.

[0011] To date, there has been very little effort to use transplantationas a potential prevention of disease due to all of the problemsassociated with transplantation as previously described. In addition, itis not yet known where transplantation can actually prevent a diseasefrom occurring other than the obvious case of whole organ failures.Moreover, for many disease states, it is not known who will beafflicted. There is some evidence that interventional transplantationcan have some preventive effect in rodents (Miller et al., J. Neurol.Immunol., 46:73-82, 1993; van Vollenhoven et al., Cell. Immunol.,115:146-155, 1988). Thus, a major role for preventive transplantationhas not been investigated.

SUMMARY OF THE INVENTION

[0012] One embodiment of the invention is a method of creatingimmunological tolerance to foreign cells, tissues or organs in a mammal,comprising the step of implanting in the mammal a tolerizing dose offoreign cells or tissue encapsulated in a biologically compatiblepermselective membrane. The method may additionally comprise the step ofadministering to the mammal a curative dose of correspondingunencapsulated cells, tissue or organ. Advantageously, the mammal is ahuman, canine or feline. Preferably, the tolerizing cells areinsulin-secreting cells; more preferably, they are pancreatic isletcells. According to one aspect of this embodiment, the membranecomprises polyethylene glycol. Preferably, the curative dose is betweenone and two orders of magnitude greater than the tolerizing dose.Advantageously, the tolerizing and curative doses are from the samespecies as the mammal. Alternatively, the tolerizing and curative dosesare from a species different from the mammal. Preferably, the tolerizingand curative doses are porcine. The method may further comprise the stepof administering one or more anti-inflammatory agents to the mammalprior to, at the same time as, or subsequent to administration of thecurative dose. Preferably, the membrane has a molecular weight cutoff ofabout 150 kDa or less. Alternatively, the membrane has a pore size ofabout 0.4 μm or less. The membrane may also have a pore size of about0.2 μm or less. Advantageously, when the tolerizing and curative dosesare from a different species, the membrane has a molecular weight cutoffof about 150 kDa or less. Preferably, the tolerizing step issubcapsular, subcutaneous, intraperitoneal or intraportal and thecurative step is intraperitoneal, intraportal or subcutaneous. Thetolerizing dose may also be administered incrementally.

[0013] The present invention also provides a method of treating diabetesin a mammal in need thereof, comprising the steps of:

[0014] implanting in the mammal a tolerizing dose of foreigninsulin-secreting cells encapsulated in a biologically compatiblepermselective membrane; then

[0015] administering to the mammal a curative dose of correspondingunencapsulated insulin-secreting cells.

[0016] Preferably, the mammal is a human, canine or feline.Advantageously, the tolerizing dose is one to two orders of magnitudeless than the curative dose. In another aspect of this preferredembodiment, the membrane comprises polyethylene glycol. Advantageously,the insulin-secreting cells are pancreatic islet cells. Preferably, themammal and the insulin-secreting cells are from the same species.Alternatively, the mammal and the insulin-secreting cells are fromdifferent species. Preferably, the tolerizing and curative doses areporcine. The method may further comprise the step of administering oneor more anti-inflammatory agents to the mammal prior to, at the sametime as, or subsequent to administration of the curative dose.Advantageously, the membrane has a molecular weight cutoff of about 150kDa or less. Alternatively, the membrane has a pore size of less thanabout 0.4 μm.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is plane view illustrating the key properties of themembrane enclosing the cells. The membrane may be configured into manydifferent device designs.

[0018]FIG. 2 is a plane view of one design of the invention, wherein twolayers of the membrane are used in a flat sheet configuration wherecells are “sandwiched” in between the two membranes and then the endsare sealed.

[0019]FIG. 3 is a tubular view of one design of the invention, whereinthe membrane is cast or rolled into a tubular configuration. The cellsare loaded in the lumen and the ends are sealed.

[0020]FIG. 4 is a spherical view of one design of the invention, whereinthe membrane is cast in a spherical configuration and cells may beencased one in each device (microcapsule) or many in a device(macrocapsule).

[0021]FIG. 5 is a graph showing blood glucose levels in mice implantedwith a tolerizing dose of 100 encapsulated NIT insulinoma aggregates.

[0022]FIG. 6 is a graph showing blood glucose levels in mice implantedwith a tolerizing dose of 50 encapsulated NIT insulinoma aggregates.

[0023]FIG. 7 is a graph showing blood glucose levels in non-tolerizedcontrol mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Goals of the Invention

[0024] The problems discussed in the foregoing Background of theInvention have previously not been solved for either micro ormacroencapsulation of cells in humans. The present invention overcomesthese problems associated with transplantation. Thus, one goal of theinvention is to eliminate the critical problems of transplantation incases where whole organ transplantation is the only alternative tocertain death. These are cases of heart or liver failure. The majoradvantage of the invention process for this application is that iteliminates the shortage of organs for the patients by making animalorgans acceptable in humans. While there are only about 4,800 humanorgan donors in the U.S. each year, the supply of animal organs fortransplant is not limited. The reason that animal organs are notpresently used is that they are acutely rejected when transplanted intohumans even with immunosuppression. Second, continuous immunosuppressionis not required in the process of the invention, thus eliminating therisk of exposing the patient to other serious diseases while the immunesystem is suppressed. Third, the cost of organ transplantation isdrastically reduced because of the unlimited supply of organs andbecause the continuous use of immunosuppressive drugs is not required.

[0025] A second goal of the invention is to make organ transplantation asafe, effective, practical therapy for those cases of disease where itis known now to be therapeutic but the risks associated with it preventits widespread therapeutic use. Examples of these disease cases arekidney failure, pancreas failure and cystic fibrosis (lung failure). Inthese cases the advantages of the process of the invention eliminate themajor obstacles. First, by making animal organs tolerated in humans the,shortage of organs for these transplant needs is solved. Second, byeliminating the need for continuous immunosuppression, these patientsare not exposed to other serious disease threats without a fullyfunctioning immune system. Third, because of plentiful organs and nocontinuous immunosuppression, the cost of this transplant procedurewould be greatly reduced.

[0026] A third goal of the invention is to make cell or tissuetransplants, as opposed to whole organ transplants, a practical therapyin cases where cells or tissue alone can cure a disease state byproviding a lacking or deficient protein, enzyme or peptide. Examples ofthese cases are insulin-secreting islet cells for Type I diabetes,Factor VIII-secreting hepatic cells for hemophilia, dopamine-secretingadrenal chromaffin cells for Parkinson's disease and collagen forarthritis. A significant advantage of the process of the invention forthese cases is that animal tissue or genetically engineered tissueexpressing an absent or deficient protein of interest can be used ifhuman tissue is scarce. In addition, cell types other than the normalprotein-secreting cells can be engineered to secrete the protein ofinterest. For example, myoblasts can be engineered by standard methodsto secrete insulin. The use of such cells is also within the scope ofthe present invention. Continuous immunosuppression is not needed toprotect the transplanted tissue and the costs would be reduced. Thus,even if pre-inoculation into the thymus with immunosuppression orirradiation of bone marrow with immunosuppression or monoclonalantibodies could be identified and produced for many disease states orencapsulation of fully therapeutic doses of tissue in some membranedevice can overcome many technical problems, the process of theinvention is a safer and more practical therapy than any of these.

[0027] A fourth goal of the invention is the treatment of autoimmunediseases including diabetes, Alzheimer's, arthritis, multiple sclerosis,myasthenia gravis and systemic lupus erythematosus. In these diseases,the body's immune system attacks and destroys one's own tissue. By usingthe process of the invention, the immune system can be induced to acceptgrafted tissue or organs to replace those that have been destroyedwithout the autoimmune destruction of the newly transplanted graft. Theadvantage of this process is that organ rejection and autoimmunedestruction are two completely different phenomena so that even withsystems that may prevent rejection, in autoimmune diseases the graftsmay still be destroyed by a different means. The process of theinvention addresses both problems.

[0028] A fifth goal of the invention is to make transplantation apractical therapy to prevent certain diseases from ever occurring, aswell as treating existing diseases as previously discussed. Theadvantage of the process that makes this possible is theimmunomodulation effect which stops or prevents the immune system fromdestruction of self tissue. Thus, for all autoimmune disorders, theprocess can be used to intervene in the course of the disease at acritical point before the immune system is initiated intoself-destruction of tissue that is necessary for normal body function.

[0029] As will be apparent from the ensuing detailed description of theinvention, the present invention meets all of these goals. Additionally,the present invention also provides a number of advantages which wouldnot have been readily apparent to one having ordinary skill in the art.

Overview

[0030] The present invention is a two step process. In the first step, asmall number of cells or tissue is implanted into a mammal inside adevice made of a biocompatible “permselective” membrane which protectsthe implanted cells from the mammal's immune system while at the sametime allowing the cells to survive. A permselective membrane is onehaving a pore size selected so that it is small enough to prevent theentry of immunological factors such as cells or antibodies, yet largeenough to allow the free passage of oxygen, nutrients and othermolecules needed to sustain the transplanted cells. In addition, themembrane pores must allow the passage of antigens which are shed fromthe transplanted cells and prevent the entry of large immune systemcells and antibodies. In a preferred embodiment, the mammal is a human.Alternatively, the mammal is a canine or feline.

[0031] One of ordinary skill in the art can readily determine the properpore size for the permselective membrane for any particular applicationof the present invention. It is preferable to use the largest pore sizepossible to prevent the entry of the undesirable elements because thelarger pores allow better diffusion of the desirable elements such asnutrients and oxygen across the membrane. Smaller pore sizes (e.g. thoseexcluding molecules greater than 100,000 daltons) are not necessarily aproblem for diffusion as has been shown in long-term survival of cellsin a 50,000 dalton membrane in vivo implant (Lacy et al., Science254:1782-1784, 1991).

[0032] Antigens shed from the transplanted cells pass through thepermselective membrane into the body of the recipient where they arefully exposed to the immune system. The immune system will recognizethese antigens as “foreign” and destroy them. This process will continuefor some time with the immune system constantly destroying the shedantigens but not able to destroy the source which is the cells protectedin the encapsulation device. In time, the immune system will begin tobecome tolerant of these antigens because they do no actual damage inthe body and the constant source cannot be destroyed. At this time, theimmune system is tolerant to that particular cell type from thatparticular donor.

[0033] Next, the second stage of the process is enacted. Now a fullytherapeutic (curative) dose of cells, tissue or whole organ from thesame donor as the tolerizing dose is implanted in the recipient for cureof the disease. Since this implant, whether cells, tissue or organ, isfrom the same donor as the small dose, it is recognized by the immunesystem as “self” and a rejection response is not elicited. The immunesystem is fully tolerant to the new implant. In one embodiment, thetolerizing dose is given as a single (bolus) dose. Alternatively, thetolerizing dose may be administered incrementally over several weeks ormonths. In a preferred embodiment, the incremental tolerizing dose isthe same as the bolus dose, only spread out in even increments. Inanother embodiment, the total incremental tolerizing dose is one tothree times the bolus tolerizing dose. As for the bolus tolerizing dose,the incremental tolerizing dose is typically one to two orders ofmagnitude lower than the curative dose.

[0034] In addition to eliminating continuous immunosuppression, thisprocess makes animal organs and cells available for human implants(xenografts). Presently, these organs or tissues are acutely rejected inhumans because of the wide immunological barriers between the species.With the process of the invention, even animal tissue will be toleratedbecause tolerance is induced gradually over time. The availability ofanimal organs for human use will save many thousands of lives each yearwhich are now lost due to the shortage of available human organs fortransplantation. In addition, this process will allow transplant therapyfor autoimmune diseases such as diabetes, arthritis, myasthenia gravisand multiple sclerosis. This is possible because as the immune system istolerized to the new tissue by the initial small implant, theself-destructive autoimmune process is suppressed. So, for diseasesrequiring organs or cellular transplants, this process eliminatescurrent shortages by making unlimited supplies of animal organs andcells available, eliminates the need for continuous immunosuppression,and protects the transplants from both rejection and autoimmunedestruction. One particularly preferred source of xenograft cells ortissue for both the tolerization and curative steps is porcine cells ortissue.

[0035] Even with the tolerizing effect of the xenograft, because of thewide species differences, an initial inflammatory reaction may occur inresponse to the curative dose. Thus, in one embodiment of the invention,the xenograft recipient is administered one or more anti-inflammatoryagents. The anti-inflammatory agent is administered either systemicallyor locally at the implantation site. The agent may be administered priorto the implant, at the time of implantation or subsequent to the implantfor a time necessary to overcome the initial inflammatory reaction. Theagents may be over-the-counter preparations such as acetaminophen oribuprofen, or a specific immunosuppressive agent such as Cyclosporine(Sandoz) or Imuran (azathioprine, Burroughs-Wellcome). The agent mayalso block the binding of a particular antigen such as CTLA4Ig (BristolMyers Squibb). The amount of anti-inflammatory agent to be administeredis typically between about 1 mg/kg and about 10 mg/kg. The extent ofinflammation will determine whether the administration of such anagent(s) is necessary. The need for such agents is only temporary andnot required for the ongoing survival and function of the transplant.

[0036] The process of the invention can also be used to prevent certaindiseases, particularly autoimmune disorders. In these cases the processis as follows. First, patients at high risk for the disease or alreadyin the very early phase of the disease are identified. At the criticaltime of the onset, the process is intervened with the small encapsulatedtissue. For example, islets are used for Type I diabetes and collagen isused for arthritis. This implant of foreign tissue immediately divertsthe attention of the immune system to the new foreign invader and itbegins the process to destroy this new threat. Because of thisdiversion, the process of self-destruction of desirable tissue that wasjust beginning is suppressed, then abandoned, then forgotten. It is, inessence, “switched off” and the damage is prevented.

Implantation of Cells to Treat Existing Diseases

[0037] The first step of this method involves acquiring small amounts ofcellular tissue for the initial tolerizing implant. The method in whichtissue is obtained depends on the type of tissue needed, the source ofthe tissue, the donor, and the amount of tissue needed. These methodsare generally well known by those skilled in the art of tissuedigestion, separation, purification, culture, and the like. Thefollowing examples are only used to illustrate that these methods arereadily available.

Islet Cells for Treatment of Diabetes

[0038] Islets are small clusters of cells located in the pancreas ofmammals. They are composed of alpha cells which make and secretesomatostatin, beta cells which make insulin, delta cells which makeglucagon and other cells which make other proteins. To isolate the isletcells which make up only 1-2% of the pancreas from the surroundingacinar tissue, the digestive enzyme collagenase is used. This process isdescribed by Ricordi (Diabetes 37:413-410, 1988, hereby incorporated byreference). Once the islets are obtained, they are purified from acinarcells and can then be implanted fresh, cultured for extended periods,cryopreserved indefinitely or encapsulated.

[0039] For use in human treatments, primary islet cells are obtainedfrom human cadaver donors or from suitable mammalian sources such asrat, cow, or pig. For use of animal tissue in humans, it is desirable toassure safety of the animal source by using specific pathogen-free (SPF)or gnotobiotic colonies or herds of animals. As an alternative to aprimary cell source, an engineered cell line which is geneticallyaltered to produce the proper regulated amounts of insulin, glucagon,somatostatin, etc. is also suitable for treatment of diabetes.

Adrenal Chromaffin Cells for Parkinson's Disease, Alzheimer's andHuntington's Disease

[0040] Adrenal chromaffin cells have multiple applications. They secretethe neurotransmitter dopamine for amelioration of Parkinson's disease,fibroblast growth factor, and can be engineered to secrete nerve growthfactor which will counter degeneration and cell death in Alzheimer's andHuntington's disease. A collagenase digestion method of isolatingadrenal chromaffin cells from the adrenal gland is described by Livett(Physiol. Rev. 64:1103-1161, 1984). Human or other mammalian sources canbe appropriate sources of this tissue.

[0041] Moreover, mammalian cells can also be genetically engineered tosecrete certain proteins or peptides whose absence or deficiency is thecause of various genetic diseases (i.e. adenosine deaminase deficiency).In addition, such cells can also be engineered to secrete variouscytokines and growth factors for the treatment of viral infections(i.e., interferon-γ) and cancer (i.e., interleukin-2). Hormonedeficiencies can also be treated by this method. Mammalian cells aretransfected with an expression vector containing a gene encoding such atherapeutic protein or peptide. These expression vectors are constructedusing standard methods well known to one of ordinary skill in the art. Atolerizing dose of these cells is encapsulated as described herein andimplanted into a mammal. Two to three weeks later, a curative dose ofthe same cells is implanted into the mammal. The cells are no longerrecognized as foreign, are not destroyed by the host immune system andcontinue to secrete the desired therapeutic protein.

[0042] Other conditions treatable by encapsulated cells producingpeptides, proteins or other therapeutic agents includehypoparathyroidism (thyroid hormone), hyperadrenocorticalism(adrenocorticotrophic factor), dwarfism (growth hormone), Gaucher'sdisease (glucocerebrosidase), Tay-Sachs (hexosaminidase A) and cysticfibrosis (cystic fibrosis transmembrane regulator). In addition, cellsexpressing stimulatory or inhibitory cytokines can be encapsulated,resulting in stimulation or inhibition, respectively, of a particularcell type. For example, erythropoietin stimulates red blood cellproduction, interleukin-2 stimulates the proliferation oftumor-infiltrating lymphocytes and interferons inhibit certain types oftumor cells. Other conditions contemplated for treatment using themethod of the present invention include amyotrophic lateral sclerosis,Alzheimer's disease, Huntington's Chorea, epilepsy, hepatitis, anxiety,stress, pain, addiction, obesity, menopause, endometriosis,osteoporosis, hypercholesterolemia, hypertension and allergies.

Other Cell Sources and Methods for Other Diseases

[0043] Other cell/tissue sources and methods include collagen recoveryfrom chicken for prevention and treatment of arthritis, Schwann cellsfrom myelin tissue for prevention and treatment of neural degenerationand Factor VIII from liver hepatocytes for treatment and prevention ofhemophilia.

[0044] The amount of cells or tissue necessary for the initialtolerizing implant will vary depending on the disease, site, source,whether the tissue is primary or immortalized and other factors.Generally, the tolerizing dose is one or two orders of magnitude lessthan a full dose implant. For example, in diabetes it usually takesbetween about 10,000-20,000 islets/kg of body weight to provide adequateinsulin production for normoglycemia. Accordingly, the initial implantdose for tolerization is about 100-2,000 islets/kilogram of body weight.Although the size of these doses are not known for all disease states,they can be optimized using routine dose/response experiments well knownto one of ordinary skill in the art. In general, between about 100cells/kg body weight and about 5,000 cells/kg body weight are suitablefor tolerization. The corresponding curative doses are between about oneand two orders of magnitude higher than these numbers.

Preparation of Encapsulation Device, Loading of Cells and Implantation

[0045] The membrane for the device is chosen for the application neededbased on its biocompatability, permeability, strength, durability,ability to be manipulated and other important considerations. A numberof materials have already been shown to be acceptable for implants inmammals. Examples of some of these materials are PAN/PVC acrylicco-polymers, hydrogels such as alginate or agarose, mixed esterscellulose, polytetrafluoroethylene (PTFE)/polypropylene (Lum et al.,Diabetes 40:1511-1516, 1991; Aebischer et al., Exp. Neurol.,111:269-275, 1991; Liu et al., Hum. Gene Ther. 4:291-301, 1993; Hill. etal., Cell Transplantation 1:168, 1992, all hereby incorporated byreference) and polyethylene glycol (PEG) conformal coatingconfigurations (U.S. Pat. No. 5,529,914, hereby incorporated byreference).

[0046] A critical factor is the pore size that can be produced in thematerial chosen. For example, PEG macromers can vary in molecular weightfrom 0.2-100 kDa. The degree of polymerization, and the size of thestarting macromers, directly affect the porosity of the resultingmembrane. Thus, the size of the macromers are selected according to thepermeability needs of the membrane. It is believed that for xenografttransplants (animal to human), antibodies of the immune system andcomplement are involved in rejection (Bachet al. TransplantationOverview 6(6):937-947, 1991). In this case, a pore size (molecularweight cutoff) of 150 kDa or smaller is desired to prevent the passageof the smallest immune antibody (IgG) through the pores of the membranecapsule. Thus, the application and its conditions will determine thechoice of membrane material from many available alternatives. Likewise,the configurations of the device will be determined by the application.For purposes of encapsulating cells and tissue in a manner whichprevents the passage of antibodies across the membrane but allowspassage of nutrients essential for cellular metabolism, the preferredstarting macromer size is in the range of about 3 kDa to 10 kDa, withthe most preferred being about 4 kDa. Smaller macromolecules result inpolymer membranes of a higher density with smaller pores.

[0047] It is also believed that for allografts (human to human), onlyentry of immune system cells must be blocked to prevent rejection oftransplanted tissue or organs (Auchincloss, Jr., TransplantationOverview 46(1):1-20, 1988. In addition, it is also desirable to excludeother cells, the smallest of which are red blood cells which have a sizeof about 7 μm. Accordingly, a membrane having a molecular weight cutoffof about 150 kDa is also suitable for encapsulation of allograft cellsor tissue because such membranes will prevent entry of such cells. In analternative embodiment, the pore size for allografts is about 0.4 μm orless to prevent the entry of immune and non-immune cells into thedevice. Cells can also extend processes (“arms”) which can enteropenings having a size of about 0.2 μm. Thus, in another preferredembodiment, the pore size is about 0.2 μm or less. In a most preferredembodiment, the pore size is as small as possible to exclude entry ofdetrimental components, but allows cell survival by permitting vitalmolecules such as nutrients, proteins and oxygen to freely pass throughthe permselective membrane. A desired pore size may be obtained byadjusting the crosslink density and length of PEG segments by one ofordinary skill in the art without undue experimentation.

[0048] If retrieval of the initial implant is unnecessary orundesirable, then a suitable configuration may be microcapsules whereonly a few or even single cells are each enclosed in separate membranes.Because of the small volume in this case, the microcapsules may simplybe injected into one of many sites for the implant. If it is desirableto retrieve or reload the device or larger numbers of cells arenecessary, a “macrocapsule” may be constructed wherein many cells areenclosed together inside one membrane. In this case, it has been shownthat the environment inside the macrocapsule may need special conditionsto allow the cells to survive. For example, an alginate matrix has beenused to immobilize islet cells and prevent their aggregation andsubsequent central necrosis (Lacy et al., Science, 254:1782-1784, 1991).

[0049] For other cell types a different environment may be needed. Themacrocapsule may be of any shape that is practical. Examples of shapescommonly used by those skilled in the art are: 1) flat sheet“sandwiches” where two layers of the membrane are top and bottom on thecells and the ends are sealed by heat welding, gluing, or other knownmeans (FIG. 2). This method provides a large surface area for membraneexposure to the host systems and generally short diffusion distanceswhich helps transport substances across the membrane; 2) A tubularmembrane formed by co-extrusion or rolling a flat sheet into a tube andsealing the ends (FIG. 3).

[0050] The cells can be placed inside the lumen at the same time themembrane is formed if co-extrusion is employed. If the tube is madefirst, the cells are loaded by syringe or other means and the ends aresealed by heat welding, gluing or other known means. As previouslydiscussed, various matrices may be employed as needed by the enclosedcells. The tubes can be any suitable length and may be joined at theends (potted) or woven if multiple tubes are used; 3) a spherical shape(FIG. 4) which has a large surface area compared to its volume and isefficient in some applications.

[0051] These are only illustrative examples of how membranes may beconfigured into devices to hold cells. One of ordinary skill in the artwill appreciate that many more configurations are possible, thusproviding great flexibility for many conceivable applications.

[0052] The loaded devices are then implanted into patients in need oftherapy. The method of implantation, site and duration are dependent onthe disease being treated. For example, in diabetes it is desirable tohave the shed antigens processed by the liver. Therefore, implantationin the peritoneum where the portal circulation would carry the antigensdirectly to the liver (intraportal) is a preferred site. Alternatively,if the dose is a small enough volume (i.e. 10 μl or less), directinjection into the portal vein is preferred. Other implantation sitesinclude under the kidney capsule and subcutaneous implantation.

[0053] For Parkinson's disease, the cells should be processed first inthe brain. Thus implanting into the interstitial region of the brain isa preferred site. For each site, the method of implantation may bedifferent. For example, intraperitoneal placement of a device fordiabetes may be performed by a minimally invasive laparoscopicprocedure. To place a device in the brain, the neurosurgeon commonlyuses stereotaxic instruments to ensure exact placement. For asubcutaneous implant, a small incision to allow a trocar to be insertedmay be used. For each preferred site, those skilled in the art willrecognize the most efficient method of implantation.

[0054] Once implanted, the cells are left in place for a period of timeduring which tolerization will occur. This time period will varydepending on the disease treated, whether an allograft or a xenografttransplant is being used, site of the implant, and other factors.Generally, tolerization requires from a few weeks to a few months.During this time, the transplanted cells constantly shed antigens fromtheir surface. These antigens comprise a variety of small moleculeswhich are constantly being replaced by living cells The antigens canpass freely out of the pores of the membrane and into the recipient atthe locations of the implant and eventually into the circulatory system.The immune system immediately recognizes these antigens as “foreign” andinitiate its mechanisms to protect the recipient from the intruder.These mechanisms are complex and not completely understood. Generally,it is believed that if the foreign matter is from a closely relatedspecies (allogeneic), cells of the immune system play the primary rolein the immunological response. These cells include T-cells, macrophages,neutrophils, and natural killer cells which seek out the source of theinvasion and destroy the foreign matter. If the foreign matter is atransplanted xenogeneic organ, preformed antibodies cause hyperacute(within minutes) reaction and rejection of the organ. If the foreignmatter is xenogeneic cells or tissue, the antigen may not be presentedand the preformed antibodies may not be the primary mechanism ofrejection. Instead, macrophages stimulate killer T-lymphocytes and later(8-10 days) antibody stimulation causes final rejection of cells ortissue.

[0055] In the present invention, however, neither system can destroy thecells of the implant when the pore size of the membrane is properlyselected for the application. For example, if allografts are destroyedby immune cells, then the membrane pores must only prevent entry ofthese cells and thus may be about 0.4 μm or smaller. Likewise, if it isnecessary to prevent antibodies from reaching the cells, the pores mustbe smaller than the smallest of the human antibodies, IgG, which is 150kDa Of course, a pore size having a molecular weight cutoff of about 150kDa or less is suitable for tolerization in both allografts andxenografts.

[0056] The use of a permselective membrane prevents the immune systemfrom destroying cells encapsulated therein, even though the immunesystem recognizes the implant tissue as foreign and mounts a classicalresponse. The immune response cannot destroy the cells because they areprotected within the membrane device. Because the immune system cannotdestroy the cells even over time, the system will come to tolerate theimplant and cease trying to destroy it. While the mechanism for thistolerization is not known, it is analogous to desensitizing patients toallergic immune reactions (i.e. antibiotics or bee stings). In fact, analternative method to the single tolerizing implant is the addition ofmore cells with more implants over time if necessary. At this point, theimmune system basically recognizes this cellular material as “self” andno longer mounts an immune response against it.

Implantation of Full Curative Dose

[0057] When the patient has been tolerized to the cells of the implant,a full curative curative size dose of the tissue or whole organ isadministered as described in the following examples.

Whole Organ Transplants—Allografts

[0058] In one embodiment, the method is used for a human allograft. Inthis embodiment, the tissue for the initial implant is taken from aliving related kidney donor by biopsy or similar method and a tolerizingdose is implanted into the patient. When the patient is tolerized, thewhole kidney is taken from the donor and transplanted into therecipient. The graft is accepted with no continuous immunosuppressionbeing necessary.

Whole Organ Transplants—Xenografts

[0059] For most embodiments, it is preferable to use animal organs forhuman transplants. In these embodiments, the procedure is as follows:suitable animal donors are identified. Sources of these donors may begenetically identical (inbred). Tolerizing cells are taken from anyanimal in the colony. Later, the whole organ is taken from any otheranimal in the colony. It is preferable that these sources are free ofall contaminants of risk to humans so they would preferably be specificpathogens free (SPF) or gnotobiotic (totally isolated in sterileconditions) colonies or herds. Heart, lungs, livers, kidneys, pancreasesand other organs may be used in this embodiment, thus eliminating thecritical shortage of these organs from the limited number of availablehuman organ donors.

Cellular Transplants—Allografts

[0060] In this embodiment, the method is used for human to humancellular transplants. A full size therapeutic dose is obtained from thecadaver donor source as previously described. For example, islet cellsare obtained from the pancreas of a human donor. The small amount neededfor the tolerizing implant is taken from the preparation, encapsulatedand implanted as previously described. The remainder of the cells arecryopreserved by known methods (Kneteman et al, Transplant. Proc.18:182-185, 1986) and are held until tolerization is completed. The fullpreparation is then thawed and ready for implantation. If, in thisembodiment, it is necessary to acquire cells from more than one donor tohave enough for a curative implant, then the cells for the initialimplant are taken from multiple donors and mixed for the implant. Therecipient is therefore tolerized to all of the cells from the multipledonors.

Cellular Transplants—Xenografts

[0061] As with whole organs, the present method allows the use ofcellular transplants from animals as well. Cells for the initial implantare taken from genetically identical animals or multiple pooled animalsas previously described. When the individual is ready for the fulltransplant, cells may be taken from any other member of the geneticallyidentical colony or from multiple pooled animals if necessary forsufficient curative quantities.

[0062] The implantation procedure for the fully curative dose of cells,whether allograft or xenograft, is dependent on the disease, thequantity of cells, the site, and other factors. For example, fordiabetes, a preferred procedure for the implantation of islet cells inhumans is to inject the cells through the portal vein so that theybecome lodged in lobes of the liver. This procedure is done under localanesthesia and is minimally invasive to the patient. For treatment ofneural disorders, cells can be implanted into any selected area of thebrain by well known stereotaxic surgical procedures. Those skilled inthe art will know preferred methods for cellular implantation for eachembodiment.

Implants for Prevention of Diseases

[0063] Identification of patient populations is dependent on the abilityto diagnose patients at high risk of developing certain diseases orthose in early stages of the disease. Rapid progress has been made inthis area of medicine primarily due to major advances in understandingand mapping the human genome. In addition, DNA amplification methods,notably the polymerase chain reaction (PCR), can be used to diagnosecertain genetic disorders. Other research areas for predicting diseasesare advancing as well.

[0064] In diabetes, the use of immune marker autoantibodies to establishpreclinical diabetes has been well studied (Palmer, Diabetes Rev.1(1):104-116, 1993). When these patients are identified, the physiciandetermines at what point in the course of the disease it would be mostadvantageous to intervene.

[0065] Individuals determined to be at risk for development of aparticular disease are implanted with the appropriate cell type asdescribed above. Methods for acquiring small amount of cellular tissuefor the initial tolerizing implant, tissue types, the amount of tissuenecessary for implantation, preparation of the encapsulation device,loading cells into the device, implanting the device into a patient,membrane parameters, device configuration, implantation methods,curative dose administration, etc. are the same as discussed hereinabovefor disease treatment.

Treatment of Diseases Arising from Lack of a Hormone

[0066] A study was performed using an insulin-producing mouse tumor cellline encapsulated in a permselective membrane coating as described inthe following example.

EXAMPLE 1 Implantation of Mouse Insulinoma Cells

[0067] The NIT insulin-producing mouse tumor cell line was encapsulatedwith PEG conformal coatings of a single, configuration, 11% PEG 4,000kDa molecular weight (See U.S. Pat. No. 5,529,914), which corresponds toa molecular weight cutoff of between about 10 kDa and about 70 kDa. Theencapsulated cells were implanted beneath the kidney capsule at twodifferent doses into C57B6 mice of a different allograft haplotype inwhich diabetes had been induced by intravenous injection (tail vein) of167 mg/kg body weight of streptozotocin (Upjohn, Kalamazoo, Mich.).Induction of diabetes by streptozotocin injection is a well knownprocedure which destroys pancreatic insulin-producing β cells.

[0068] Tolerizing doses of encapsulated insulinoma cells were 50 or 100cell aggregates, each containing about 1,000 cells. Encapsulated cellswere implanted beneath the kidney capsule using standard surgicalprocedures. Curative implants of unencapsulated insulinoma cells(2,000-3,000 insulinoma cell aggregates, each containing about 1,000cells) were administered by free intraperitoneal injection 15 or 20 daysafter the tolerizing dose to determine whether a sufficient quantity ofcells survived. Control animals were given only the curative dose ofinsulinoma cells. Blood glucose levels were monitored and are shown forthe 100 encapsulated NIT aggregate tolerizing dose, 50 encapsulated NITaggregate tolerizing dose and non-tolerized controls (FIGS. 5, 6 and 7,respectively).

[0069] The severity of streptozotocin-induced diabetes in these micecaused several of the animals to die during the periods of observationand during procedures done as part of the study. Table 1 indicates thenumber of animals involved in the study and their outcomes. The degreeof diabetes is very high, with values over 500 mg/dl (shown as 500) forall streptozotocin-induced animals in the study. Many of these severelydiabetic animals died of their diabetes during the study or following aprocedure as noted. As shown in FIG. 5, of the first group of 8 diabeticmice receiving 100 encapsulated aggregates, only four survived for thechallenge 20 days later with the unencapsulated aggregates. Two of thesedied overnight following the IP injection. The remaining two recipientsboth had a sudden and marked reduction in their glucose values between 5and 9 days, with glucose values reaching levels of 40 mg/dl and below(BM5 and BM11, FIG. 5 and Table 1). If the insulin-secreting insulinomacells induce immunological tolerance, the curative implant will berecognized as “self” and will not be destroyed by the recipient's immunesystem. Because the NIT cells are tumor cells which double every 2-3days in vitro, their survival would be expected to result in recipienthypoglycemia due to the increasing insulin-producing cell mass thatwould occur from living and growing tumor cells.

[0070] In the second group of three recipients of 50 encapsulatedaggregates for 15 days, two died of their diabetes prior to challengewith unencapsulated NIT cells. The one animal that received thechallenge of unencapsulated NIT cells (BM16) has not exhibited anyreduction in blood glucose values for the same time of observation (FIG.6). None of the control animals only challenged with unencapsulated NITcells exhibited any reduction in blood glucose values (FIG. 7).

[0071] The results indicate that encapsulated NIT cells given as a smallmass prior to a large, unencapsulated curative cell implant permits thesecond curative dose to survive, reducing blood glucose values in apattern suggestive of NIT tumor cell growth. A smaller dose ofencapsulated NIT cells did not give this result. Control animals thatonly received unencapsulated NIT cells in a curative dose exhibited noreduction in blood glucose. These results indicate that the preliminaryencapsulated implant tolerized the host to the following unencapsulatedcurative dose. When such a preliminary encapsulated implant was notdone, the curative unencapsulated implants had no effect on bloodglucose and were presumably destroyed by the host. TABLE 1 # Encap.Toler. Cell Delay Un- # Encap. Agg. to Cure encap. Unenc. Effect onAnimal Im- Tol. Im- Cell Cell Agg. Blood # plant Dose plant Implant CureGlucose BM1 yes 100 20 days yes 2348 none-died* BM3 yes 100 20 daysno-died — n/a BM4 yes 100 19 days no-died — n/a BM5 yes 100 20 days yes2348 down to 40 BM6 yes 100 20 days no-died — n/a BM7 yes 100 20 daysyes 2348 none-died* BM10 yes 100 20 days no-died — n/a BM11 yes 100 19days yes 2348 down to 40 BM14 yes 50 15 days no-died — n/a BM15 yes 5015 days no-died — n/a BM16 yes 50 15 days yes 2348 none-500 BM29 no 0 —yes 2352 none-500 BM31 no 0 — yes 2352 none-500 BM32 no 0 — yes 2352none-500 BM34 no 0 — yes 2352 none-500 BM35 no 0 — yes 2352 none-died*BM36 no 0 — yes 2352 none-500 BM40 no 0 — yes 2352 none-500 BM41 no 0 —yes 2352 none-died* BM42 no 0 — yes 2352 none-500 BM43 no 0 — yes 2352none-died* BM44 no 0 — yes 2352 none-500 BM45 no 0 — yes 2352 none-500

EXAMPLE 2 Use of Encapsulated Islets for Induction of AllograftTolerance in Rats

[0072] Rat pancreatic islet cells are isolated by a standard collagenasedigestion method (Ricordi, Diabetes 37:413-410, 1988) and cultured forthree days prior to PEG encapsulation. Donor islets are derived from theWistar Furth (WF) strain having MHC haplotype RT1-U. Recipients are ofthe Lewis strain having MHC haplotype RT1-1. Transplants across thisstrain combination are normally rejected within three weeks. Islettransplant mass is dosed on the basis of a standard 150 μm diameter ratislet; an Islet Equivalent (Ieq). Islets are quantified and tested forsterility and mycoplasma prior to encapsulation and implantation.

[0073] Islet cells are conformally coated with 11% PEG 4,000 kDamolecular weight by the method described in U.S. Pat. No. 5,529,914. Asa negative control, acellular cross-linked dextran beads areencapsulated in a similar manner. Diabetes is induced in fasted Lewisrats by intravenous injection of streptozotocin (65 mg/kg) one weekprior to implantation of the tolerizing dose and monitored during thatweek for blood glucose levels and weight changes. Rats are considereddiabetic once their blood glucose level exceeds 350 mg/dl. Rats having aminimal weight loss and blood glucose levels of 300-350 mg/dl are usedfor the study.

[0074] Diabetic rats are implanted by trochar with a subcutaneous 30 daytime release depot insulin (Linplant, Lishin, Ontario, Canada) to reducethe chances of ketosis/acidosis and to stabilize their diabetes. Animalsremain hyperglycemic at this Linplant dose (2 units of bovine insulin in24 hours—lasts 30 days).

[0075] Diabetic MHC disparate Lewis rats are surgically implanted oncewith encapsulated donor WF islets at the renal subcapsular site afteranesthetization. The dose of implanted cells varies as outlined in Table2. TABLE 2 Group N Dose Rationale 1 12 1200 encap islets high dosesensitization/ tolerization 2 12  600 encap islets low dose tolerization3 12  300 encap islets very low dose tolerization 4 12 1200 encapacellular beads control for polymer

[0076] As a control, a set of recipients (Group 4) is implanted withencapsulated acellular beads to control for possible polymer effects intolerization. All implanted animals are maintained for intervals asshown in Table 3 prior to the second transplantation. At the time ofimplantation, serum samples from each animal are drawn and retained forfuture immunological analysis. TABLE 3 Implant Interval Group N Dose(days) Rationale 1a 4 1200 encap islets 30 high dose sensitize/tolerize-short interval 1b 4 1200 encap islets 60 high dose sensitize/tolerize-intermediate interval 1c 4 1200 encap islets 90 high dosesensitize/ tolerize-long interval 2a 4  600 encap islets 30 low dosetolerization- short interval 2b 4  600 encap islets 60 low dosetolerization- intermediate interval 2c 4  600 encap islets 90 low dosetolerization- long interval 3a 4  300 encap islets 30 very low dosetolerization- short interval 3b 4  300 encap islets 60 very low dosetolerization- intermediate interval 3c 4  300 encap islets 90 very lowdose tolerization- long interval 4a 4 1200 encap acell-beads 30 polymercontrol- short interval 4b 4 1200 encap acell-beads 60 polymer control-intermediate interval 4c 4 1200 encap acell-beads 90 polymer control-long interval

[0077] During the indicated period, animals are monitored for weightchanges and blood glucose levels. One week before the second transplant,one animal in each of Groups 1a-1c, 2a-2c, 3a-3c and 4a-4c is sacrificedand the implant site analyzed by histological methods for determiningviability of the tolerizing cells.

[0078] Lewis rats remaining in Groups 1-4 receive a second transplant(curative dose) of WF islets which are unencapsulated. Transplant sitesin each animal are intraportal (IP) at a dose of 6,000 Ieq and at onekidney with a dose of 100 Ieq (See Table 4). 6,000 Ieq implanted intothe liver is known to be a curative dose in the rat diabetes model. The100 Ieq kidney capsule implant is only for histology at the end of theexperiment. At the time of the second implant, serum samples from eachanimal are drawn and retained for future immunological analysis. For thenext three weeks, animals are monitored for blood glucose levels andweight changes. At the termination of the experiment, graft sites areprocessed for histology. At this time, serum samples from each animalare again drawn and retained for future immunological analysis. TABLE 4Recipient Dose Implant Duration of Group N haplotype # of islets SitesTransplant 1a 3 RT1-1 6000 IP/kidney 3 weeks 1b 3 RT1-1 6000 IP/kidney 3weeks 1c 3 RT1-1 6000 IP/kidney 3 weeks 2a 3 RT1-1 6000 IP/kidney 3weeks 2b 3 RT1-1 6000 IP/kidney 3 weeks 2c 3 RT1-1 6000 IP/kidney 3weeks 3a 3 RT1-1 6000 IP/kidney 3 weeks 3b 3 RT1-1 6000 IP/kidney 3weeks 3c 3 RT1-1 6000 IP/kidney 3 weeks 4a 3 RT1-1 6000 IP/kidney 3weeks 4b 3 RT1-1 6000 IP/kidney 3 weeks 4c 3 RT1-1 6000 IP/kidney 3weeks

[0079] In Groups 1 and 4, no changes in the diabetic state are measured.In Group 4, rejection occurs in the expected two week time frame asmeasured by a transient normoglycemia followed by a return to thediabetic state. In Group I, a more rapid rejection of the implant due tosensitization of the recipients occurs. In the recipients previouslyexposed to tolerizing doses of encapsulated WF islets (Groups 2 and 3),islet cells survive and result in a continuous maintenance ofnormoglycemia.

EXAMPLE 3 Use of Encapsulated Islets for Induction of AllograftTolerance in Humans

[0080] Human islets are isolated from cadavers and 1,500 islets/kg bodyweight are PEG-encapsulated and implanted under the kidney capsule in adiabetic patient. After two months, a curative dose of 15,000unencapsulated islets/kg body weight are injected intraportally. Insulinadministration is continued during the course of the protocol up toadministration of the curative dose. Blood glucose levels are constantlymonitored and are within the normal range.

EXAMPLE 4 Treatment of Parkinson's Disease (Xenograft)

[0081] Adrenal chromaffin cells are isolated from inbred baboon adrenalglands and 1,000 cell/kg body weight are encapsulated in an appropriatePEG conformal coating. The capsule is implanted into the interstitialbrain region of a human by a neurosurgeon using stereotaxic instruments.After 1 month of tolerization, 10,000 unencapsulated cells/kg bodyweight are injected into the same brain region. Significant improvementin the condition is observed.

EXAMPLE 5 Prevention of Hemophilia

[0082] A male individual at risk of developing hemophilia, an x-linkeddisorder, by virtue of family history, is subjected to genetic screeningto determine the presence or absence of the gene encoding Factor VIII,and to clotting time analysis. If the gene is absent or clotting time isreduced, 2,500 liver cells/kg recipient body weight are isolated from ahuman donor and encapsulated in a PEG conformal coating. Theencapsulated cells are implanted under the kidney capsule. One monthlater, 5,000 cryopreserved liver cells/kg recipient body weight (fromthe same donor) are injected intraportally. Clotting time issignificantly improved.

EXAMPLE 6 Liver Transplant (Xenograft)

[0083] An individual in need of a liver transplant is subcutaneouslyimplanted with 1,000 PEG-encapsulated liver cells/kg body weightisolated from an inbred baboon. Two months later, the entire liver istransplanted into the individual. Signs of organ rejection and vitalsigns are monitored over several months. Rejection does not occur.

EXAMPLE 7 Prevention of Myasthenia Gravis (Xenograft)

[0084] Myasthenia gravis is an autoimmune disorder resulting from thepresence of antibodies against the acetylcholine receptor on neurons. Anindividual having very early signs of the disease is implanted under thekidney capsule with a tolerizing dose of 2,500 PEG-encapsulated neuralcells/kg recipient body weight expressing the acetylcholine receptorisolated from baboons. This results in tolerization to the acetylcholinereceptor and prevention of the disorder.

[0085] It should be noted that the present invention is not limited toonly those embodiments described in the Detailed Description. Anyembodiment which retains the spirit of the present invention should beconsidered to be within its scope. However, the invention is onlylimited by the scope of the following claims.

What is claimed is:
 1. A method of treating diabetes in a mammal in needthereof, comprising the steps of: implanting in said mammal a tolerizingdose of insulin-secreting cells from the same species as said mammalencapsulated in a biologically compatible permselective membrane; thenadministering to said mammal a curative dose of correspondingunencapsulated insulin-secreting cells.
 2. The method of claim 1,wherein said mammal is a human, canine or feline.
 3. The method of claim1, wherein said tolerizing dose is one to two orders of magnitude lessthan said curative dose.
 4. The method of claim 1, wherein saidinsulin-secreting cells are pancreatic islet cells.
 5. The method ofclaim 1, wherein said membrane comprises polyethylene glycol.
 6. Themethod of claim 1, wherein said tolerizing and curative doses areporcine.
 7. The method of claim 1, further comprising the step ofadministering one or more anti-inflammatory agents to said mammal priorto, at the same time as, or subsequent to administration of saidcurative dose.
 8. The method of claim 1, wherein said membrane has amolecular weight cutoff of about 150 kDa or less.
 9. The method of claim1, wherein said membrane has a pore size of less than about 0.4 μm. 10.The method of claim 9, wherein said membrane has a pore size of lessthan about 0.2 μm.
 11. The method of claim 1, wherein said curative doseis between one and two orders of magniture higher that said tolerizingdose.
 12. The method of claim 1, wherein said implanting step issubcapsular, subcutaneous, intraperitoneal or intraportal.
 13. Themethod of claim 1, wherein said administering step is intraperitoneal,intraportal or subcutaneous.
 14. The method of claim 1, wherein saidtolerizing dose is administered incrementally.